This Competitive Revision proposal requests funding to support new research aims that are outside of the scope of the approved parent grant. The original grant focuses on loss-o-function studies to define the roles of Eya and Six genes in early otic placode induction and inner ear morphogenesis and understand the developmental and molecular pathogenesis of inner ear defects that occur in the Branchio-Oto-Renal (BOR) syndrome. This revision application proposes to use gain-of-function approaches to define the role of Eya1 in regulating hair cell differentiation program, which is not proposed in the parent grant. In the mammalian auditory system, degeneration of the sensory hair cells is the most common cause of hearing loss affecting approximately 10% of the worldwide population. A potential approach to restore normal hearing is to use stem/progenitor cells because of their intrinsic capacity for regeneration. However, it is currently unclear whether there are stem/progenitor cells capable of regenerating hair cells present in the cochlea. The auditory sensory hair cells are derived from progenitor cells specified in the prosensory domain within the cochlea epithelium at ~E13.5-14.5. Recent studies have shown that Sox2 is required for the specification of sensory cell lineage, while Atoh1 has been shown to be a key regulator for hair cell development but is not required for specifying prosensory progenitors. To date, however, how exactly the sensory cell lineage is specified and how the hair cell fate is determined are still poorly understood. Furthermore, how Atoh1 expression is activated and the molecular genetic pathways regulating hair cell differentiation program are essentially unknown. Our recent data suggest that Eya1 may interact with Sox2 to not only specify sensory progenitors but also regulate hair cell commitment possibly by activating Atoh1 expression. The goal of this Competitive Revision application is to investigate the intrinsic programs of sensory progenitors, focusing on the evolutionarily conserved Eya1-Six1 transcription complex, which is critical for normal sensory organ formation in the inner ear, via gain-of-function approaches. Thus, this revision application in combination with the loss of function studies proposed in the parent grant will greatly strengthen our analysis of Eya1-Six1 transcription complex during sensory hair cell differentiation and provide a deeper understanding of how Eya1-Six1 acts to regulate hair cell differentiation program and new insights into the basis for hearing loss that occurs in human BOR patients. The proposed revision application will not only stimulate the economy by enabling hiring one full-time post-doc and one full-time research technician to accomplish the new studies but also generate novel resources, which will make significant contribution to the field of hair cell development, regeneration and maintenance. PUBLIC HEALTH RELEVANCE: This revision application proposes to investigate the intrinsic programs of sensory progenitors, focusing on the evolutionarily conserved Six1-Eya1 transcription complex, which is critical for normal sensory organ formation in the inner ear and pathogenesis of human Branchio-Oto-Renal syndrome (BOR). Identifying genes and proteins and understanding their roles and functions in the development of sensory cells may eventually lead to prevention or regeneration of inner ear hair cells.